The development of new drugs has contributed to the progress that has been made in recent years in the treatment of various types of cancer. However, certain cancers are refractory to the chemotherapeutic agents that are currently in use. These malignancies tend to be particularly virulent and are associated with a high mortality rate. Most existing chemotherapeutic agents have undesirable side effects as well. Consequently, there is ongoing interest, both within the medical community and among the general population, in the development of novel chemotherapeutic agents for the treatment of malignant tumors and other types of cancer.
One naturally occurring ribonuclease isolated from the leopard frog (Rana pipiens) has shown antitumor activity in patients with advanced inoperable pancreatic cancer and malignant mesothelioma in PhaseI/II studies (Delmonte, Oncology Times Vol. 18, No. 8, August 1996). The antitumor activity of this ribonuclease (referred to as Onconase) has been examined in more than 350 patients with a variety of solid tumors and has been shown to have antitumor activity against metastatic pancreatic carcinoma, advanced metastatic breast cancer, and malignant mesothelioma. Onconase is now being used Phase III human clinical trials against pancreatic and liver cancer.
In general, Onconase appears to be relatively safe to use as a chemotherapeutic in humans. It does not cause any of the major toxicities associated with conventional cytotoxic drugs, such as myelosuppression, gastrointestinal toxicity, or mucositis. Approximately one third of patients treated with Onconase developed flu-like symptoms and Grade 3 arthralgia with or without peripheral edema. The most serious complication associated with administration of Onconase is decreased renal function, which was observed in Phase I dose-ranging trials. The decreased renal function was reversible upon dose reduction.
In addition to treating various types of cancers, ribonucleases may have utility in the treatment of persons infected with HIV. Noncytotoxic concentrations of Onconase have been reported to significantly inhibit HIV production in several human cell lines persistently infected with HIV.
The cytotoxicity of ribonucleases was first demonstrated in solid tumors injected with milligram quantities of bovine pancreatic ribonuclease A (RNase A; EC3.1.27.5) (Ledoux, L. Nature 176:36-37, 1955; Ledoux, L. Nature 175:258-259). Smaller doses were found to have no effect on the tumors (de Lamirande, Nature 192:52-54, 1961). A ribonuclease that is cytotoxic at low levels was discovered in bull seminal plasma (Floridi et al., Ital. Biol Sper. 43:32-36, 1967; Dostal et al., J. Reprod. Fertil. 33:263-274, 1973). A ribonuclease with even greater cytotoxicity was isolated from frog eggs (Ardelt et al., J. Biol. Chem. 266:245-251, 1991), and this ribonuclease is the one now being tested in clinical trials under the name Onconase.
Ribonucleases catalyze the degradation of RNA. It has been demonstrated that the ribonucleolytic activity of cytotoxic ribonucleases is required for cytotoxicity (Kim et al., J. Biol. Chem. 270:10525-10530, 1995; (Ardelt et al., J. Biol. Chem. 266:245-251, 1991). Although the cytotoxicity of ribonucleases requires ribonucleolytic activity, ribonucleolytic activity alone is not sufficient to explain differential cytotoxicities observed among ribonucleases. For example, the ribonucleolytic activity of RNase A is approximately 1000-fold greater than that of Onconase, but Onconase has much greater cytotoxicity than RNase A. Thus, other properties of the enzymes must account for this difference.
Vertebrate cells contain a ribonuclease inhibitor (RI) that protects the cells from the potentially lethal effects of ribonuclease. The RI is a 50-kDa cytosolic protein that binds to ribonucleases with varying affinity. For example, RI binds to members of the bovine pancreatic ribonuclease A (RNase A) superfamily of ribonucleases with inhibition constants that span ten orders of magnitude, with K.sub.i s ranging from 10.sup.-6 to 10.sup.-16
The cytotoxicity of a ribonuclease appears to be inversely related to the strength of the interaction between RI and the ribonuclease. For example, RNase A, which binds RI with a high affinity (K.sub.i =10.sup.-14 M) is not cytotoxic. In contrast, Onconase binds RI with relatively low affinity (K.sub.i.gtoreq.10.sup.-6 M).
Some natural ribonucleases do not bind to RI. Bovine seminal ribonuclease (BS-RNase) is 80Cr identical in amino acid sequence to RNase A, but unlike RNase A, BS-RNase exists in a dimeric form. It has been shown that the quaternary structure of BS-RNase prevents binding by RI, thereby allowing the enzyme to retain its ribonucleolytic activity in the presence of RI (Kim et al., Biochem. J. 308:547-550, 1995; Kim et al., J. Biol. Chem. 270:10525-10530, 1995; Kim et al., J. Biol. Chem. 270:31097-31102, 1995). Onconase, a 104 amino acid residue protein that shares a high degree of homology with RNase A, is nevertheless resistant to binding by RI. The RI-Onconase complex has a K.sub.d of .gtoreq.10.sup.-6 M (Boix et al., J. Mol. Biol. 257:992-1007, 1996), which is at least one hundred million times less than that of the RI-RNase A complex.
Thus, the key distinction between Onconase and RNase A that accounts for the differential cytotoxicity observed in these ribonucleases is that Onconase is resistant to inhibition by RI. Normal cells produce an RI that binds ribonucleases noncovalently with a 1:1 stoichiometry and inhibits ribonucleolytic activity. The lower binding affinity of Onconase for RI prevents effective inhibition of the ribonucleolytic activity. This is the best explanation for the observed fact that Onconase is cytotoxic while ribonuclease A is not.
Recent studies in which the plasma clearance and tissue distribution of Onconase and RNase A in mice were examined showed that at three hours after the injection of Onconase or RNase A, 57% of Onconase is found in the kidney, whereas only 0.9% of human pancreatic ribonuclease is found in the kidney (Sung, et al. Cancer Res. 56:4180, 1996). The decreased renal function observed in patients who receive Onconase may be a consequence of an inability to effectively clear the Onconase protein from the kidneys.
A preferred therapeutic ribonuclease would be a cytotoxic ribonuclease that can be cleared from the kidneys more readily than Onconase. A cytotoxic ribonuclease that is readily cleared from the kidneys would be less likely to cause renal toxicity. Because reduced renal function is dose-limiting for Onconase, a cytotoxic ribonuclease that does not interfere with renal function would potentially offer the advantage of greater flexibility in determining optimal dosages. A ribonuclease with lower toxicity could be administered at higher doses where indicated, e.g., for those cases in which increased dosages would afford a more effective treatment for particular types of cancers or for particular individuals.
The side effects experienced by participants in clinical trials of Onconase are symptoms that are commonly associated with immune reactions. It is reasonable to expect that a ribonuclease from a species more closely related to humans than is the leopard frog would be less likely to cause an immune reaction. Less likely still to evoke an immune response would be a human ribonuclease. The intensity of an immune reaction may also be greater when larger amounts of the immunogenic protein are administered. Therefore, a cytotoxic ribonuclease with a higher specific activity than that of Onconase may potentially be a more effective chemotherapeutic. It may be possible to achieve effective cytotoxicity with the administration of smaller amounts of protein, thereby reducing the incidence and severity of symptoms associated with an immune reaction.
New cytotoxic ribonucleases with antitumor activity are needed to enhance the spectrum of chemotherapeutics available for treatment of human cancers.